In 1996, Susan Rosenberg, then a young professor at the University of Alberta, undertook a risky and laborious experiment. Her team painstakingly screened hundreds of thousands of bacterial colonies grown under different conditions, filling the halls outside her lab with tens of thousands of plates of bacteria. “It stank,” Rosenberg recalled with a laugh. “My colleagues hated me.”

The biologist, now at Baylor College of Medicine in Houston, hoped to resolve a major debate that had rocked biology in different incarnations for more than 100 years. Were organisms capable of altering themselves to meet the needs of their environment, as Jean Baptiste Lamarck had proposed in the early 1800s? Or did mutations occur randomly, creating a mixture of harmful, harmless or beneficial outcomes, which in turn fueled the trial-and-error process of natural selection, as Charles Darwin proposed in “On the Origin of Species”?

Although Darwin’s ideas have clearly triumphed in modern biology, hints of a more Lamarckian style of inheritance have continued to surface. Rosenberg’s experiments were inspired by a controversial study, published in the late 1980s, that suggested that bacteria could somehow direct their evolution, “choosing which mutations will occur,” the authors wrote — a modern molecular biologist’s version of Lamarckian theory.

Baylor College of Medicine

Susan Rosenberg, a biologist at Baylor College of Medicine in Houston, studies how bacteria mutate when under stress.

Rosenberg’s results, published in 1997, disputed those findings, as other’s had before, but with a twist. Rather than targeting specific traits, as Lamarck’s theory would have predicted, the mutations struck random genes, with some good outcomes and some bad. However, the process wasn’t completely random. Rosenberg’s findings suggested that bacteria were capable of increasing their mutation rates, which might in turn produce strains capable of surviving new conditions.

“Cells are able to adapt to stress not by knowing exactly what they need to do, but by throwing the dice as a population and making random changes to the genome,” said James Broach, a biologist at Pennsylvania State University’s College of Medicine in Hershey who studies a similar phenomenon in yeast. “That will allow stressed progeny to find an escape route.”

Rosenberg expected the biology community to be relieved. Darwin, after all, had prevailed. But some scientists questioned the findings. Indeed, the research triggered debates that played out in the pages of scientific journals for several years. Accurately measuring mutation rates can be tricky, and given that most mutations are harmful to the cell, boosting their frequency seemed like a risky evolutionary move.

Over the past decade, however, labs around the world have found similar patterns in bacteria, human cancer cells and plants. And Rosenberg and others have pinpointed the molecular mechanisms underlying the stress-induced mutations, which vary from organism to organism.

Phil Hastings, Baylor College of Medicine

Bacterial colonies mutate more frequently when put under stress, as shown by the visible mutation in these blue-green colonies.

Scientists are now beginning to explore how these mechanisms can be targeted for medical treatments, such as new cancer therapies and long-lasting antibiotics. The research provides insight into how both cancer cells and pathogenic bacteria evolve resistance to treatment, a stubborn and deadly problem that has plagued physicians and drug developers.

Last March, for example, Ivan Matic, a research director at the French National Institute of Health and Medical Research in Paris, and collaborators found that very low levels of antibiotics, present in the water supply from human and animal waste, were enough to push bacteria into a stressed state, boosting their mutation rates. “It’s a spectacular example of stress-induced mutation,” said Matic, whose findings were published in Nature Communications last year. Some of these mutations made the bacteria resistant to antibiotics, suggesting that exposure to a low dose of one antibiotic could prime bacteria to evolve resistance to other antibiotics as well.

Most scientists now accept that stress boosts mutation rates in some organisms, although questions remain regarding how much the phenomenon contributes to their evolution. “What’s controversial now is whether cells evolved to do this to create mutations,” said Patricia Foster, a biologist at Indiana University in Bloomington.

Molecular Mistakes

In 1943, Max Delbrück and Salvador Luria, two of the founding fathers of molecular biology, performed a landmark experiment designed to examine the nature of mutation. They showed that mutations in bacteria arise spontaneously, rather than in response to a specific environmental pressure. The work, which ultimately won them a Nobel Prize, was all the more impressive given that scientists did not yet know the structure of DNA.

We now know that mutations arise in a variety of ways, typically when a cell is copying or repairing its DNA. Every so often, the molecular machinery that makes DNA inserts the wrong building block, or the copying machinery jumps elsewhere in the genome and copies the wrong piece. Those changes can have no effect, or they can alter the structure of the protein that the DNA produces, changing its function for better or, more often, for worse.

Rocky Mountain Laboratories, NIAID, NIH

Under stressful conditions, such as when food is scarce, E. coli bacteria employ an enzyme that tends to make mistakes when copying DNA.

According to Delbrück and Luria’s model, these accidents happen randomly, gradually accruing over time. But scientists have occasionally considered an alternative — that organisms can in some cases control how they mutate, enabling them to more rapidly evolve to adapt to new environments. Indeed, in unpublished correspondence, Delbrück’s one-time adviser, Wolfgang Pauli, questioned whether the mutation process could be truly random. As Rosenberg, who read the letters at a conference in 2007, recalls, Pauli argued that “a simple probabilistic model would not be sufficient to generate the fantastic diversity we see.”

The debate surfaced again in 1988, when the biologist John Cairns and collaborators at Harvard University made a provocative proposal in the journal Nature, that bacteria could somehow choose which genes to mutate. The evidence? Bacteria incapable of digesting a sugar called lactose evolved that ability when given no other alternative food. “The paper was hugely controversial,” recalled Foster, a friend of Cairns’ who collaborated with him on follow-up studies. “Letters flew back and forth.”

The idea that cells can regulate their mutation rates is not as outlandish as it might seem. Certain immune system cells, for example, mutate much more frequently than others, enabling them to produce varieties of antibodies that can subdue novel invaders. But these cells are confined to the immune system and do not pass along their mutations to the next generation.

It was Cairns’ finding that inspired Rosenberg to undertake her experiments. She suspected that his proposal was wrong, but not entirely. “People fought about it for five years in the front pages of major journals,” she said. “It was clear to me that it was a hugely important question.”

Subsequent research from both Rosenberg and Foster showed that mutations were scattered across the E. coli genome, rather than directed to specific genes, as Cairns had proposed. (Cairns abandoned his hypothesis after follow-up experiments with Foster.) They also found that stress, including lack of food, was a crucial factor in boosting the mutation rate.

“It was a surprise for people,” Rosenberg said. “Cells actually decide to turn up their mutation rate when they are poorly adapted to the environment. That’s a different kind of picture from constant random mutation that is blind to the environment the cell is in.”

Mamun et al., Science 2012

A network of more than 90 proteins is required to trigger stress-induced mutations in E. coli bacteria.

With a background in molecular biology rather than evolution, Rosenberg felt that the only way to quell remaining skepticism was to figure out how the mutations were forming. She and Foster independently spent the next few years hammering away at the molecular mechanism underlying the change in mutation frequency in E. coli. Under normal conditions, the bacteria employ an enzyme that carefully copies DNA. But Rosenberg and Foster found that when bacteria are under stress, a mistake-prone enzyme takes over, bumping up the frequency of mutations.

Some scientists are still skeptical, if not about the phenomenon itself, then about how significant it is for an organism’s survival and evolution. At the heart of the debate is a paradox. Most random mutations will be harmful to the organism, knocking out vital proteins, for example. Therefore, more frequent mutations would be likely to generate a less-fit population. “People have still been doubting the phenomenon because they believe that it would be maladaptive,” said Foster. “Increasing the mutation rate would increase deleterious mutations as well as advantageous ones.” Some scientists think that evolution would not select such a mechanism, she said.

However, results of modeling experiments designed to mimic real-world conditions suggest that boosting the mutation rate during times of stress is beneficial both to individual cells and the overall population, even if beneficial mutations occur only rarely. “We think it’s built into the system as a means of the cells hedging their bets based on the future conditions,” Broach said. Individual cells use different strategies for dealing with stress, he said,but “because [the cells] are related, the gene pool is going to survive.”

Another question is whether stress-induced mutations are unique to lab-grown E. coli or are found in many biological systems. But a flurry of studies on other microbes, including those in the wild, and other organisms suggest that stress-induced mutations are a widespread phenomenon.

Beyond Bacteria

Soon after Rosenberg published her initial experiments in bacteria in the 1990s, Peter Glazer, a physician and biologist at Yale University School of Medicine, began making similar discoveries in cancer. When tumors grow quickly, some cancer cells lack adequate blood flow, which restricts access to oxygen and puts them under considerable stress. Glazer found that similar to the way starving or otherwise stressed bacterial cells bump up their mutation rates, cancer cells deprived of oxygen undergo more frequent mutations. Over the past decade, Glazer has narrowed in on the mechanism for this phenomenon — the molecular processes that normally repair faulty DNA are suppressed.

Although the different systems studied to date employ different mechanisms for dealing with stress, “many have the result that [mutation frequency] increases,” Matic said. Stress-induced mutations in the pathogenic bacteria that Matic studies, as well as in yeast and cancer cells, result from different mechanisms than in Rosenberg’s E. coli.

More recently, Glazer and collaborators have begun to think about how to apply their findings to cancer treatment. One of the challenges in treating cancer is finding drugs that kill tumor cells but leave healthy cells alone. If certain DNA repair pathways are altered in cancer cells, you can start looking for drug candidates that target mechanisms unique to cancer, Glazer said. Moreover, like bacteria, tumors often evolve resistance to new drugs. To address both cancer and antibiotic resistance, researchers could try to prevent stress-induced mutation in cancer cells or bacteria in the first place.

Clare McLean/UW Medicine

Christine Queitsch, a biologist at the University of Washington in Seattle, has shown that even plants mutate more frequently under certain conditions.

Both Rosenberg and Christine Queitsch, a biologist at the University of Washington in Seattle, said that medical researchers should consider the molecular mechanisms driving certain clinical phenomena, such as antibiotic resistance or drug resistance in cancer, when developing new treatments. “In the clinic, we typically treat the outcomes of evolutionary processes, for example by cutting the cancer out,” Queitsch said. “Wouldn’t it be cool if we could take away the cancer cell’s ability to evolve?”

Bacteria, yeast and even cancer cells to a certain extent are rapidly growing single-celled organisms. Is stress-induced mutation limited to those lowly cells, or does it also apply to more complex, multicellular organisms? The phenomenonis more difficult to study in plants and animals for a variety of reasons, including because they have longer lifespans. But new evidence suggests that stress can boost the occurrence of mutations in multicellular organisms as well.

In 2012, Queitsch found that she could drive up mutations in plants by dampening the function of a protein called HSP90, which helps other proteins fold properly — or assume their functional form — especially under times of stress. When cells are stressed, more proteins need HSP90 to fold properly, and they compete for the resource. In that scenario, DNA repair proteins may fold incorrectly, hampering the repair process and leading to more mutations.

Blind Fish

Nicolas Rohner

A protein called HSP90 may have played a role in the loss of eyes in a species of cave-dwelling fish, bottom, called Astyanax mexicanus, which evolved from surface-dwelling ancestors, top.

New genetic mutations aren’t the only way for organisms to adapt to changes in their environment. A growing body of evidence suggests that organisms can harbor mutations whose effects are masked until times of stress. The mediator is a protein called HSP90, which helps proteins fold so that they can function properly. According to Susan Lindquist, a biologist at the Whitehead Institute for Biomedical Research in Cambridge, Mass., this protein under normal conditions helps even mutated proteins fold. But under stressful conditions, more proteins need help from HSP90, and there isn’t enough to go around. The result is that the misfolded proteins can no longer function properly, which triggers a range of strange outcomes, such as square eyes or shriveled wings in fruit flies. “All organisms are near the precipice of a protein-folding crisis,” Lindquist said.

In a December paper published in Science, a group of researchers — including Lindquist and Clifford Tabin, a biologist at Harvard Medical School — found that HSP90 likely played a role in a species of cave-dwelling fish losing their eyes. According to the scientists’ model, the abrupt change in conditions that occurred when the fish’s surface-dwelling ancestors were trapped in caves taxed the HSP90 system. That stress revealed that some of the fish had existing mutations giving them smaller eyes, which proved beneficial to living in dark caves. Studying surface-dwelling cousins of the cave fish in the lab, the researchers found that both reducing HSP90 concentration and altering water conditions, as might have occurred during the transition to caves, triggered substantial variation in eye size.

It’s likely that the same thing is happening in other organisms, Queitsch said, because HSP90 is highly conserved, meaning that organisms ranging from plants to flies, worms and people have almost exactly the same protein.

Stress-induced mutations have now been observed in a variety of organisms, “but the question is whether they help the organism adapt,” said Jan Drake, a biologist who recently retired as head of the Spontaneous Mutation & DNA Repair Group at the National Institute of Environmental Health Sciences. According to Drake, the evidence in single-cell organisms is strong. One example is Matic’s stressed bacteria that evolve resistance to antibiotics. But it’s an open question in plants and animals, he said.

Accident or Evolution Machine?

For scientists developing new drugs, understanding exactly why cancer cells mutate more frequently under stress doesn’t really matter. Simply understanding how it works — and how to stop it — is likely to improve treatments. But for others, including Rosenberg and Matic, the driving force behind the phenomenon represents one of the most interesting aspects of stress-induced mutation. “Most molecular biologists ask how it works, but I am in the minority in asking the question why,” Rosenberg said. “I think those are the most important and fascinating questions.”

Two basic possibilities exist: Cells could have evolved this mechanism as a sort of evolution-boosting machine, triggering excess mutations precisely when conditions call for new traits. The alternative argument is that the phenomenon is a byproduct of molecular mechanisms that evolved for other reasons, such as the need to repair DNA damage when under stress. If that’s the case, error-prone enzymes — and the higher mutation rate they produce — are simply the price the cell must pay.

It’s difficult to experimentally prove what provoked the development of a specific trait, but both Rosenberg and Matic strongly believe that nature evolved this mechanism on purpose. “I am convinced that under certain conditions, high mutation rates can be adaptive for bacteria, but that’s still an open question in the community,” Matic said. He has shown that the mechanism for generating mutations under stress is highly regulated, suggesting that it exists for a purpose, not as a byproduct of other forces.

Foster said she believes both interpretations are true. She suggested that cells might have initially evolved different DNA repair mechanisms under stress to improve the cells’ survival. The higher mutation rates that resulted could have been “an accident that was maintained in the system because of adaptive value,” she said.

To try to further Rosenberg’s case that stress-induced mutation evolved for a purpose, her team explored the network of genes that are required for the phenomenon to occur. The results, published in Science in 2012, identify more than 90 proteins that are required for the process. More than half are involved in either sensing stress or turning on stress responses, which Rosenberg said supports the idea that the cell has purposely structured its mutation mechanism to respond to stress.

After two decades of work in the field, “I now view evolution as more responsive, plastic and trainable or ‘smart’ than I did previously,” Rosenberg said. “I think that others have also had their view shifted in this direction.”

The early (Archean) evolution of unicellular stress responses which enhance
mutation rates in general seems like a good strategy, and self-evident. The
increase in deleterious mutations will inevitably be offset by those which
confer survival and success, especially in microbes having astronomical
population numbers… nature is profligate. It also is tempting to posit
that such a stress response strategy will itself become more widespread
and varied, over deep time. And it could help answer questions about
how complex new innovations could occur, seemingly de novo. I also
intuit that along the entire genome of a microbe, say, there are probably
hot spots, where due to chemistry or local architecture, mutations are
significantly more likely. Perhaps the stress responses discussed in this
article might also act on such regions more strongly?

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